Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for delivery of nucleic acids (polynucleotides and oligonucleotides) to cells, the process is one step in reaching a goal of providing therapeutic processes. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polyion complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective.
In terms of intravenous injection, polynucleotides must cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter from 75–150 nm. The trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20–30 nm. The size of the DNA complexes is also important for the cellular uptake process. After binding to the target cells the DNA-polycation complex should be taken up by endocytosis.
Inhibition of gene expression can be affected by antisense polynucleotides, siRNA mediated RNA interference and ribozymes. RNA interference (RNAi) describes the phenomenon whereby the presence of double-stranded RNA (dsRNA) of sequence that is identical or highly similar to a target gene results in the degradation of messenger RNA (mRNA) transcribed from that target gene (Sharp 2001). It has been shown that dsRNAs <30 bp in length (short interfering RNAs or siRNAs) delivered to a cell, induce RNAi in mammalian cells in culture and in vivo (Tuschl et al. 1999; Elbashir et al. 2001). There are two major approaches to initiate siRNA-mediated silencing in mammalian cells. First, synthetic siRNA duplexes (typically between 19–30 base pairs in length) can be designed and generated against any gene the sequence of which is known. The synthetic siRNA then has to be delivered into the cytoplasm. Second, expression cassettes that will generate siRNA within the cell can be delivered to the cell. The two basic types of siRNA expression constructs code either for a hairpin RNA containing both the sense and the antisense sequence, separated by a loop region, or they contain two separate promoters driving the transcription of the sense and antisense RNA strand separately.
The intravascular delivery of nucleic acid has been shown to be highly effective for gene transfer into tissue in vivo (U.S. application Ser. No. 09/330,909, U.S. Pat. No. 6,627,616). Non-viral vectors are inherently safer than viral vectors, have a reduced immune response induction and have significantly lower cost of production. Furthermore, a much lower risk of transforming activity is associated with non-viral polynucleotides than with viruses.
Applicants have provided a process for delivering a compound across the endothelial barrier to the extravascular space and then to a cell.